• Highly recurring mutations are present in WM, including MYD88 L265P, warts, hypogammaglobulinemia, infection, and myelokathexis-syndrome–like mutations in CXCR4, and ARID1A.

  • Small, previously undetected CNAs affecting B-cell regulatory genes are highly prevalent in WM.

The genetic basis for Waldenström macroglobulinemia (WM) remains to be clarified. Although 6q losses are commonly present, recurring gene losses in this region remain to be defined. We therefore performed whole genome sequencing (WGS) in 30 WM patients, which included germline/tumor sequencing for 10 patients. Validated somatic mutations occurring in >10% of patients included MYD88, CXCR4, and ARID1A that were present in 90%, 27%, and 17% of patients, respectively, and included the activating mutation L265P in MYD88 and warts, hypogammaglobulinemia, infection, and myelokathexis-syndrome–like mutations in CXCR4 that previously have only been described in the germline. WGS also delineated copy number alterations (CNAs) and structural variants in the 10 paired patients. The CXCR4 and CNA findings were validated in independent expansion cohorts of 147 and 30 WM patients, respectively. Validated gene losses due to CNAs involved PRDM2 (93%), BTG1 (87%), HIVEP2 (77%), MKLN1 (77%), PLEKHG1 (70%), LYN (60%), ARID1B (50%), and FOXP1 (37%). Losses in PLEKHG1, HIVEP2, ARID1B, and BCLAF1 constituted the most common deletions within chromosome 6. Although no recurrent translocations were observed, in 2 patients deletions in 6q corresponded with translocation events. These studies evidence highly recurring somatic events, and provide a genomic basis for understanding the pathogenesis of WM.

Waldenström macroglobulinemia (WM) is a distinct indolent B-cell malignancy in the World Health Organization classification. WM shares many clinical and pathological features with other B-cell lymphomas and multiple myeloma, which often complicates the diagnosis of this entity. Immunoglobulin M (IgM) monoclonal gammopathy of unknown significance (MGUS) is a precursor state for WM. Approximately 2% of IgM MGUS patients evolve to a B-cell malignancy per year, with most of these individuals progressing to WM.1,-3  Since the initial description of WM by Jan Waldenström in 1944, the genetic basis for WM has been elusive. We therefore employed whole genome sequencing (WGS) utilizing tumor-derived DNA from 30 patients, which included paired germline/tumor sequencing for 10 patients. The initial findings of this study resulted in the identification of a recurrent somatic mutation, L265P in MYD88, in over 90% of WM patients that distinguished WM from other overlapping entities such as marginal zone lymphoma, chronic lymphocytic leukemia (CLL), and multiple myeloma, wherein MYD88 L265P was either absent or infrequently observed (<10%).4,-6  Importantly, MYD88 L265P was present in IgM MGUS with a frequency of 50% to 80% using allele-specific polymerase chain reaction (AS-PCR), suggesting an early oncogenic event for this mutation and that other genomic events are likely to be responsible for WM disease progression. Activating MYD88 mutations are not unique to WM, and appear in other B-cell lymphomas including primary central nervous system lymphoma (50%) and activated B-cell (ABC) subtype diffuse large B-cell lymphoma (DLBCL) (8% to 29%).7,-9  A multi-hit model would also help explain why no significant differences in treatment response, progression free, and overall survival based solely on MYD88 mutation status determination were observed in a large series of WM patients.10 

Little is known about the genetic structural variant (SV) changes such as copy number alterations (CNAs) or translocations in WM. By fluorescence in situ hybridization and array comparative genomic hybridization, deletions in chromosome 6q21-23 have been identified in 40% to 60% of WM patients, with concordant gains in 6p in 41% of those with 6q deletions.11,-13  Gains in chromosomes 3q, 4, 18, 8q, and Xq, as well as losses of 11q23, 13q14, and 17p have also been described in up to 20% of WM cases.14,15  We therefore sought to more fully characterize the molecular events responsible for WM pathogenesis using WGS.

Sample collection and preparation

Bone marrow (BM) aspirates and peripheral blood (PB) samples were collected in heparinized syringes from 30 patients with the clinicopathological diagnosis of WM, as defined by the Second International Workshop on WM.16  Participants provided informed consent prior to sample collection, in accordance with the Dana-Farber Cancer Institute Institutional Review Board. This study was conducted in accordance with the Declaration of Helsinki. Relevant clinical characteristics and blood work are presented in supplemental Table 1 on the Blood Web site. BM and PB mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Paque (Amersham-Pharmacia Biotech, Piscataway, NJ). WM lymphoplasmacytic cells (LPC) were isolated from the BM mononuclear cells by CD19+ selection using immunomagnetic MACS micro-beads (Miltenyi Biotech, Auburn, CA). To minimize potential contamination from circulating WM LPC, PBMCs were depleted of CD19+ cells using CD19+ MACS micro-beads.

Complete methodology for the WGS analysis and validation is available in the supplemental Methods.

Thirty patients who met the International Consensus Criteria for WM diagnosis were evaluated in this study.16  The clinical characteristics were typical of WM of this cohort and are presented in supplemental Table 1. Tumor DNA from CD19+ BM LPC was isolated from the 30 patients, along with DNA from CD19-depleted PBMCs for use in germline analysis. All 30 tumor and 10 PBMC samples were submitted for WGS with Complete Genomics, as previously described.4 

Coding variant analysis

WGS findings were filtered for novel nonsynonymous variants as described in the supplemental Methods. This identified 5835 variants in 4427 genes. The 10-paired genomes were used to identify genes containing at least one somatic variant. Variant data from all 30 WM tumor genomes were then fit to this model wherein each affected gene was categorized as potentially somatic, germline, or unclassified if variants were only observed within the 20 unpaired samples (supplemental Table 2). Genes were further categorized by mutational frequency and validated based on bioinformatic strength and biological relevance, using Sanger sequencing of both tumor and germline DNA (Table 1). These cut-off scores were significantly lower than our previous analysis, allowing for the detection of a number of additional mutations. Primers and representative chromatograms are provided in supplemental Table 3. A full list of somatic and germline mutations affecting genes in the COSMIC cancer gene census are shown in supplemental Tables 4 and 5, respectively.17 

As previously described, a somatic T/C mutation in MYD88 resulting in an L265P substitution was observed in 27 of 30 samples. In addition to the L/P-activating substitution in the primary transcript, this mutation resulted in a stop loss in two of the alternative isoforms. The potential functional significance for these shorter transcripts in MYD88 L265P is not known. A subclonal C/G mutation was found in MYD88 resulting in an amino acid substitution (S219C) in 1 patient who also had the L265P mutation.8  No other mutations in MYD88 were observed in the study population.

To better understand the MYD88 wild-type (WT) population, we identified somatic mutations found exclusively in the 3 MYD88 WT patients. Cross-listing these genes with COSMIC, we observed that 2 of the 3 (67%) patients had damaging mutations in MLL2, while mutations in the genes JAK2, PRCC, SUZ12, BCL10, KDM6A, and SETD2 were each observed once. Sanger sequencing confirmed the somatic presence of MLL2 mutations in these patients as previously described.4  Although a larger sample size is needed to determine if the high frequency of MLL2 mutations is typical of the MYD88 WT WM population, it nonetheless represents a gene of great interest since MLL2 mutations are highly recurrent in follicular lymphoma and DLBCL patients.18 

The next most common somatic mutation was observed in the G-protein coupled receptor, CXCR4. CXCR4 is a chemokine receptor that promotes WM survival, migration, and adhesion to the BM stroma through interactions with its ligand, CXCL12.19  Eight of 30 (27%) patients harbored 1 of 5 somatic variants in CXCR4, each of which were identical or functionally similar to mutations associated with WHIM syndrome (Figure 1).20,21  WHIM syndrome is a rare, autosomal dominant genetic disorder that is caused by frameshift or nonsense mutations in the carboxyl-terminal cytoplasmic tail (c-tail) of CXCR4. These mutations occur after the last of the seven transmembrane helices, destroying the c-tail but leaving the regions responsible for ligand binding and downstream signaling via g-proteins intact.22  These c-tail mutations result in the loss of regulatory serines resulting in impaired internalization and prolonged activation.23,-25  To validate this finding, we screened an independent cohort of 147 WM patients for CXCR4 mutations by Sanger sequencing. Combined with the 30 patients in the WGS cohort whose results were validated by Sanger sequencing, 51 of 177 (28.8%) patients had c-terminal mutations in CXCR4. Of these 177 patients, 160 demonstrated the MYD88 L265P mutation. Importantly, 50 of 51 (98%) patients with CXCR4 c-terminal mutations were also MYD88 L265P positive. Conversely, 16 of 17 (94.1%) of the MYD88 WT patients were WT for CXCR4 (P = .026).

Figure 1

Somatic CXCR4 mutations in WM are similar to those found in WHIM syndrome. Somatic CXCR4 nonsense and frameshift mutations found in 177 WM patient samples. (A) Protein sequence for the canonical full-length transcript (NP_003458.1) demonstrates that similar to the variants responsible for WHIM syndrome, these mutations result in a truncation of the cytosolic tail containing the regulatory phospho-serines leaving the seven transmembrane helix region involved in signaling and ligand binding intact. (B) The crystal structure of homodimeric CXCR4 with the carboxyl terminal tail highlighted in yellow and indicated by the white arrows. (C) Precise location and number of WHIM-like somatic mutations in WM at transcript level and detailed mutation-type summary of the most frequently mutated amino acid, S338.

Figure 1

Somatic CXCR4 mutations in WM are similar to those found in WHIM syndrome. Somatic CXCR4 nonsense and frameshift mutations found in 177 WM patient samples. (A) Protein sequence for the canonical full-length transcript (NP_003458.1) demonstrates that similar to the variants responsible for WHIM syndrome, these mutations result in a truncation of the cytosolic tail containing the regulatory phospho-serines leaving the seven transmembrane helix region involved in signaling and ligand binding intact. (B) The crystal structure of homodimeric CXCR4 with the carboxyl terminal tail highlighted in yellow and indicated by the white arrows. (C) Precise location and number of WHIM-like somatic mutations in WM at transcript level and detailed mutation-type summary of the most frequently mutated amino acid, S338.

Close modal

Other genes with validated somatic mutations in this series of patient samples included: ARID1A (5 of 30; 17%), CD79B (2 of 30; 7%), TP53 (2 of 30; 7%), MYBBP1A (2 of 30; 7%), MUC16 (1 of 30; 3%), TRAF2 (1 of 30; 3%), TRAF3 (1 of 30; 3%), RAG2 (1 of 30; 3%), and NOTCH2 (1 of 30; 3%) (Figure 2).

Figure 2

Summary of study results. (A) Copy number results per patient for the independent 30-patient validation cohort as determined by quantitative polymerase chain reaction (qPCR). Somatic MYD88 L265P and WHIM-like CXCR4 mutations were assessed in this population and annotated here for reference. The eight CNA targets selected for validation were chosen based on the 10-paired patient WGS analysis and technical validation studies. (B) Validated mutations in the 30-patient WGS cohort. Somatic status of all findings was confirmed by germline Sanger sequencing. (C) Overall frequency of validated somatic mutation (top) or CNA (bottom) for the independent 30-patient WGS and validation cohorts, respectively.

Figure 2

Summary of study results. (A) Copy number results per patient for the independent 30-patient validation cohort as determined by quantitative polymerase chain reaction (qPCR). Somatic MYD88 L265P and WHIM-like CXCR4 mutations were assessed in this population and annotated here for reference. The eight CNA targets selected for validation were chosen based on the 10-paired patient WGS analysis and technical validation studies. (B) Validated mutations in the 30-patient WGS cohort. Somatic status of all findings was confirmed by germline Sanger sequencing. (C) Overall frequency of validated somatic mutation (top) or CNA (bottom) for the independent 30-patient WGS and validation cohorts, respectively.

Close modal

SV analysis

SVs were inferred from high confidence discordant mate-pair and relative coverage data supplied by Complete Genomics. To gain further insight into the SV events underlying WM, we combined individual patient data for lesser allele fraction (LAF), heterozygous call rate, normalized relative coverage, somatic CNAs, and somatic SV calculations with cohort-wide small variant distribution and CNA regions of statistical significance (supplemental Figure 1). These analyses aid in the detection of complex events such as acquired uniparental disomies (aUPD), which can be observed in chromosomes 3p and 21 in supplemental Figure 1. The most common aUPD was at 3p, occurring in 4 of 30 (13%) patients and whose location and size were highly conserved between the samples (Figure 3). This locus included the MYD88 L265P locus, resulting in homozygous somatic mutation in these patients. Additional large aUPDs were observed on chromosomes 1, 2, 5, 9, 17, 21, and X, with many of these affecting the validated small variant findings (Table 1).

Figure 3

aUPD in chromosome 3p in 4 patients with WM. (A) Representative excerpts from paired tumor and germline tissue documenting an aUPD on chromosome 3 for a single WM patient. The red histogram below the ideogram corresponds to the heterozygous call rate. Below that, copy number (black line) and normalized relative coverage (orange) is shown above the LAF measurements (blue). LAF values more than 2 standard deviations from the mean are shown in green. (B) Sanger sequencing of MYD88 L265P for this patient with a near homozygous mutant signal. (C) Heterozygous call rate, LAF, and relative coverage for the 4 patients with aUPDs in chromosome 3p demonstrating the near identical location of the aUPD for the 4 affected patients.

Figure 3

aUPD in chromosome 3p in 4 patients with WM. (A) Representative excerpts from paired tumor and germline tissue documenting an aUPD on chromosome 3 for a single WM patient. The red histogram below the ideogram corresponds to the heterozygous call rate. Below that, copy number (black line) and normalized relative coverage (orange) is shown above the LAF measurements (blue). LAF values more than 2 standard deviations from the mean are shown in green. (B) Sanger sequencing of MYD88 L265P for this patient with a near homozygous mutant signal. (C) Heterozygous call rate, LAF, and relative coverage for the 4 patients with aUPDs in chromosome 3p demonstrating the near identical location of the aUPD for the 4 affected patients.

Close modal

Translocations were rare events, and we were unable to demonstrate any recurrent translocations in the 10 paired samples (supplemental Table 5). One patient had two distinct t(2;17) translocations disrupting both alleles of RNF213. A t(6;X) translocation disrupting the gene BIA3 was responsible for the chromosome 6q deletion observed in another patient (supplemental Figure 1). A third patient had a chromothriptic event centered on chromosome 6, which made up over half of all translocations observed in this study (supplemental Figure 2), and contributed to 6q deletions observed in the patient’s tumor cells. In this patient, we performed PCR for t(6;7) predicted to disrupt BMP5 and ANKRD7, as well as t(6;11) predicted to disrupt GRIA4 and PKHD1, respectively, and validated both of these findings. For 2 of 5 patients with paired normal/tumor genomes and in whom 6q deletions were identified, translocations were responsible for these regional losses. Macro-level analysis of normalized coverage for all 30 WM samples demonstrated that large deletions in 6q were present in 13 of 30 (43%) cases with corresponding gains in 6p in 3 of 13 (23%) cases. The next most common alteration observed was an amplification of chromosome 4, which occurred in 7 of 30 (23%) patients, confirming previous fluorescence in situ hybridization and array comparative genomic hybridization findings.14,15 

Small somatic CNAs

To detect small somatic deletions and amplifications, we calculated 100 kb blocks of the log-transformed ratio of tumor-to-germline relative coverage in each of the paired genomes. Significant CNAs were then compared across all 10 patients in order to identify statistically significant regions. CNAs consisted mostly of isolated 100 kb blocks both within and outside of chromosome 6 (supplemental Table 6 and supplemental Figure 1E). To investigate the biological relevance of these nonchromosome 6 CNAs, we performed functional enrichment analysis on the gene set affected by these alterations. Unique functional annotation results are listed in order of significance in Figure 4A.

Figure 4

Characterization of somatic CNAs in WM. (A) Functional annotation for genes affected by CNA found outside of chromosome 6. The list is ordered by statistical significance and filtered only for duplicated functional annotations matching more than one category. (B) Deletions of matching size were randomly distributed across the genome in 10 000 trials. The number of affected total RefSeq and COSMIC genes was calculated for each group. Results represent mean values with empirical 95% confidence intervals.

Figure 4

Characterization of somatic CNAs in WM. (A) Functional annotation for genes affected by CNA found outside of chromosome 6. The list is ordered by statistical significance and filtered only for duplicated functional annotations matching more than one category. (B) Deletions of matching size were randomly distributed across the genome in 10 000 trials. The number of affected total RefSeq and COSMIC genes was calculated for each group. Results represent mean values with empirical 95% confidence intervals.

Close modal

To better characterize these highly recurrent deletions, consecutive blocks of equal significance were merged together to form 194 statistically significant regions with a median length of 100 kb (range = 100 kb to 4900 kb). Of these regions, 134 (69%) were found outside of chromosome 6 with a median length of 100 kb (range = 100 kb to 300 kb), of which 126 of 134 (94%) were restricted to a single 100 kb region. Within chromosome 6, CNAs were larger with only 30 of 60 (50%) CNAs constituting single 100 kb regions (P = 7.709 × 10−12). CNAs targeted COSMIC census genes for 14 of 172 (8.14%) genes outside of chromosome 6 vs 4 of 239 (1.67%) genes within chromosome 6, respectively (P = .0024). To determine the probability of generating these results as a random by-product of genomic instability, we calculated the number of total genes affected by these deletions (N = 411) and the number of these genes found in the COSMIC database (N = 18). We randomly distributed matching deletion sets across the genome, and enumerated the total number of overlapping genes and the number of those genes listed in the COSMIC census (Figure 4B). These simulations revealed a statistically significant increase in targeting of COSMIC genes by CNAs outside of chromosome 6. Affected genes in the COSMIC census were: BTG1 (9 of 10; 90%), FOXP1 (7 of 10; 70%), FNBP1 (7 of 10; 70%), CD74 (7 of 10; 70%), TOP1 (6 of 10; 60%), MYB (5 of 10; 50%), CBLB (5 of 10; 50%), ETV6 (5 of 10; 50%), TNFAIP3 (5 of 10; 50%), FBXW7 (5 of 10; 50%), PRDM1 (5 of 10; 50%), TFE3 (4 of 10; 40%), JAK1 (4 of 10; 40%), MAML2 (4 of 10; 40%), FAM46C (4 of 10; 40%), EBF1 (4 of 10; 40%), STL (4 of 10; 40%), and BIRC3 (4 of 10; 40%). Other affected genes of interest included PRDM2 (8 of 10; 80%), HIVEP2 (8 of 10; 80%), ARID1B (7 of 10; 70%), as well as LYN (7 of 10; 70%).

There were no singular regions of statistical significance in 6q. Some patients had multifocal deletions in this region suggesting multiple target genes (Figure 5). Neither of the previously suspected target genes for 6q loss (ie, PRDM1 and TNFAIP3) were included in the regions of highest statistical significance.11,12  The relative coverage data from all 10-paired patients is shown in Figure 5B, illustrating that only 3 patients had highly clonal deletions in 6q. Two additional patients had subclonal deletions in 6q; therefore, 5 of 10 (50%) paired patients had at least subclonal loss. Deletions in HIVEP2 (8 of 10; 80%), as well as ARID1B (7 of 10; 70%) and BCLAF1 (7 of 10; 70%) constituted the most common deletions in chromosome 6, and were present in patients with and without visible 6q losses.

Figure 5

Somatic deletions identified on chromosome 6 by WGS in WM patients. (A) Frequency of statistically significant chromosome 6 deletions from the 10 paired patients highlighting genes of interest. The positions of the deletions are mapped against chromosome 6 cytogenetic bands. (B) Relative coverage across chromosome 6 for each of the 10 paired samples. Losses in 6q were not always single contiguous deletions, and some patients had deleted segments restricted to a subclone. The frequency of deletions for genes including HIVEP2 and ARID1B were higher than the corresponding number of large block deletions.

Figure 5

Somatic deletions identified on chromosome 6 by WGS in WM patients. (A) Frequency of statistically significant chromosome 6 deletions from the 10 paired patients highlighting genes of interest. The positions of the deletions are mapped against chromosome 6 cytogenetic bands. (B) Relative coverage across chromosome 6 for each of the 10 paired samples. Losses in 6q were not always single contiguous deletions, and some patients had deleted segments restricted to a subclone. The frequency of deletions for genes including HIVEP2 and ARID1B were higher than the corresponding number of large block deletions.

Close modal

To test our validation system and examine the accuracy of the guanine-cytosine-normalized relative coverage data, we used a PCR-based copy number assay to validate the established 6q22 deletion in a subset of patients (Figure 6A). Having recapitulated the expected results, we conducted additional validation studies for 8 of our top targets in the same 5 patients using commercially available assays. To establish the frequency of these CNAs, we validated these 8 findings in an independent cohort of 30 WM patients revealing the following somatic losses: PDRM2 (28 of 30; 93%) at 1p36.21, BTG1 (26 of 30; 87%) in Chr. 12q21.33, HIVEP2 (23 of 30; 77%) at 6q24.2, MKLN1 (23 of 30; 77%) at 7q32, PLEKHG1 (21 of 30; 70%) at 6q25.1, LYN (18 of 30; 60%) at 8q12.1, ARID1B (15 of 30; 50%) at 6q25.1, and FOXP1 (11 of 30; 37%) at 3p13 (Figure 2). For MKLN1 and HIVEP2, germline CNAs were significant, and therefore, tumor-to-germline relative coverage should be interpreted accordingly (Figure 6C). While many results appeared subclonal, we observed a strong correlation between the PCR-relative copy number and WGS coverage predictions (Figure 6D).

Figure 6

Validation results using qPCR for the most frequent somatic deletions identified by WGS in WM patients. Five patient samples, 3 from the paired and 2 from the unpaired WGS cohorts, were selected for validation studies using qPCR copy number assays. All assays were run in at least triplicate. Results represent median values and ranges. (A) Validation of deletion in the known 6q deletion in 2 patients at HINT3 (6q22.32). (B) Representative validation results normalized to germline as determined by qPCR. Deletions deemed significant by Welch’s t test are denoted by asterisk (*). (C) Somatic relative copy number needs to be interpreted in context. Significant germline copy number variation was noted in both HIVEP2 and MKLN1. (D) Comparison of whole genome and qPCR validation estimates of relative somatic copy number demonstrating similar clonal estimates.

Figure 6

Validation results using qPCR for the most frequent somatic deletions identified by WGS in WM patients. Five patient samples, 3 from the paired and 2 from the unpaired WGS cohorts, were selected for validation studies using qPCR copy number assays. All assays were run in at least triplicate. Results represent median values and ranges. (A) Validation of deletion in the known 6q deletion in 2 patients at HINT3 (6q22.32). (B) Representative validation results normalized to germline as determined by qPCR. Deletions deemed significant by Welch’s t test are denoted by asterisk (*). (C) Somatic relative copy number needs to be interpreted in context. Significant germline copy number variation was noted in both HIVEP2 and MKLN1. (D) Comparison of whole genome and qPCR validation estimates of relative somatic copy number demonstrating similar clonal estimates.

Close modal

CXCR4 and MYD88 L265P mutation status was determined in BM samples for all 30 patients by Sanger sequencing and AS-PCR, respectively. There were fewer median validated deletions in CXCR4-mutated patients (5) compared with WT (7; P = .002), and with the median total number of 6q CNAs (2) compared with WT (3; P = .007) (Figure 2). This was also true for any combination of two of the three validated 6q genes, thereby ruling out possible biasing by a single gene (P < .023 for all).

The most pronounced finding from this study was the discovery of a somatic mutation in MYD88 (L265P), which was present in 90% of patients with WM. The details of this discovery were previously published following the preliminary examination of our WGS results.4  MYD88 serves as an adaptor molecule in Toll-like receptor (TLR) and interleukin-1 receptor signaling, mediating interleukin-1 receptor-associated kinase 4 (IRAK4) and IRAK1 activation, and downstream signaling through nuclear factor κB (NFκB).4,8,26  Mutations in MYD88 are significant given the importance of NFκB signaling in WM cell growth and survival.27  We recently demonstrated that MYD88 L265P can trigger NFκB signaling through an IRAK-independent pathway by direct interaction with Bruton’s tyrosine kinase (BTK) in WM cells, suggesting parallel pathways for NFκB activation.28  In contrast to the findings by Ngo et al who observed multiple MYD88 mutations (L265P, V217F, S219C, M232T, S243N, and T294P) in ABC-subtyped DLBCL patients, our findings were limited to the identification of L265P and S219C as a subclonal event in 1 WM patient with a L265P mutation.8  We also did not observe mutations in CARD11 in WM patients, demonstrating differences in potential oncogenic drivers and dependence on TLR signaling for WM vs ABC-subtyped DLBCL.

The next most common somatic variant after MYD88 L265P was in CXCR4, which was present in 27% of the patients. The mutations identified in WM tumor cells recapitulated those found in the germline of patients with WHIM syndrome. To our knowledge, this is the first time WHIM-like mutations have ever appeared as somatic mutations, and also be associated with a malignant disease. In WHIM syndrome, the loss of regulatory serines in the c-tail of CXCR4 are known to impair receptor internalization, thereby prolonging G-protein and β-arrestin signaling.25  CXCR4 stimulation by its ligand CXCL12 is known to activate AKT1 and mitogen-activated protein kinase family signaling, as well as facilitate cell migration and homing in WM cells.19  The prolonged activation of CXCR4 signaling due to WHIM mutations may therefore exaggerate these effects in WM cells, and deserves further study. Nearly all patients with CXCR4 mutations had also carried the MYD88 L265P mutation, and comprehensive studies will be required to delineate the relative impact of these mutations on WM clinical and treatment response characteristics. The presence of CXCR4 mutations may also offer a targeted approach to therapy of WM by use of CXCR4 antagonists given their successful record in the treatment of WHIM syndrome patients.29  Several antagonists to CXCR4 have been developed and are in clinical trials, including plerixafor, BMS-936564, AMD-070, TG-0054, and others, and warrant investigation alone and/or in combination in WM patients.

Whereas most WM cases in our series did not have mutations in CXCR4, it is possible that CXCR4 signaling may still be critical for many of these patients and offer an opportunity for targeted therapy with CXCR4 antagonists.21  In WM, polymorphisms of the CXCR4 ligand, CXCL12, have been associated with poor posttreatment clinical outcomes.30  Copy loss of RGS17 that was observed in 5 of 10 (50%) patients in our series may also affect this pathway. CXCR4 signals downstream through Giα G-proteins, increasing phosphorylated AKT1 levels and promoting cell survival, while RGS17 inhibits Giα G-proteins by promoting guanosine triphosphate hydrolysis.31,32  In an analogous case, RGS17 inactivation was observed in ovarian cancer affecting Giα G-protein coupled receptor downstream signaling in response to lysophosphatidic acid.31 

The other major pathway identified in this study was the loss of chromatin remodeling proteins, ARID1A and ARID1B.33 ARID1A was the third most common single nucleotide variant target in WM, with 5 of 30 (17%) patients having validated nonsense or frameshift mutations. In 1 patient, a mutation in ARID1A (Y551 frameshift) was homozygous as a result of an aUPD, whereas in another patient a nonsense mutation (Q2037*) was opposite CNA loss, resulting in biallelic inactivation (Table 1). Loss of the alternate family member ARID1B was present in 7 of 10 (70%) of the paired patient samples, making it a more frequent 6q target than either PRDM1 or TNFAIP3 (5 of 10; 50% for both).33  One potential target for this pathway was the B-cell development regulator EBF1, which itself was affected by copy number variation deletions in 4 of 10 (40%) patients.34,35  Both ARID1A and ARID1B are members of the switch/sucrose nonfermentable family of proteins that are known to be mutated in other neoplastic malignancies, wherein they are thought to exert their effects via p53 and CDKN1A regulation.36,-38 TP53 itself was mutated in 2 of 30 (7%) of the sequenced genomes, including 1 case of biallelic mutation. Both PRDM2 and TOP1 participated in TP53-related signaling and were deleted in 8 of 10 (80%; 28 of 30 in validation) and 6 of 10 (60%) patients, respectively.39,40  Together, these findings imply that multiple mutations exist in the genome of WM patients which dysregulate the DNA damage response.

The loss of additional cancer-associated genes was observed including ETV6, which was deleted in 6 of 10 (60%) samples analyzed. Deletions of ETV6 have been observed in acute lymphoblastic leukemia (ALL), acute myeloid leukemia, and myelodysplastic syndromes.41,-43 ETV6 is a member of the E-twenty six transcription factor family and acts primarily as a transcriptional repressor involved in hematopoiesis, and functions as a tumor suppressor in myeloid leukemias.44 FOXO3, which was deleted in 6 of 10 (60%) patients, has been shown to negatively regulate growth and survival in mantle cell lymphoma, B-CLL, and natural killer cell neoplasms.45,-47  In B-CLL, phosphorylation of FOXO3 by AKT1 downstream of CXCL12 and CXCR4 prevents FOXO3 nuclear translocation and signaling.46  Upon nuclear translocation, FOXO3 can induce transcription of BTG1, which was deleted in 9 of 10 (90%; 26 of 30 in validation) paired patients.48  While FOXO3 (6q21) was not explicitly validated, 6q deletions were significantly less common in CXCR4-mutated patients and there was a trend for fewer BTG1 deletions as well. BTG1 is a nuclear coactivator that modulates transcription, a member of the antiproliferative TOB/BTG protein family, and was recently shown to be recurrently mutated in several studies of DLBCL.18,49,50  Small deletions affecting BTG1 have been reported in up to 12% of the B-cell precursor subtype of ALL.43,51,52 BTG1 deletions in B-cell precursor subtype of ALL were noted to coincide with deletions in ETV6 and EBF1, both of which were recurrently deleted in our study and suggests that these events may be functionally related.52  Preclinical studies of BTG1 in ALL have shown that the loss of BTG1 is associated with glucocorticoid resistance. These findings may explain the poor responses observed to single-agent steroids in WM, and warrant further investigation of BTG1 loss in treatment outcomes in patients undergoing glucocorticoid-inclusive therapy.53,54  Finally, biallelic loss of RNF213 resulting from two distinct t(2;17)s was observed in 1 of 10 paired patients, and represents an interesting finding since RNF213 is a fusion partner of anaplastic lymphoma kinase and MYC in acute anaplastic large cell lymphoma. In the WM patient with the t(2;17) translocation, RNF213 was translocated to the intergenic space indicating that the loss of RNF213 itself may play an important role in oncogenesis.55,56  It is important to note that most CNAs were consistent with heterozygous loss, and classical tumor suppressors require biallelic loss for oncogenesis. However, these losses could lead to pathway modulation due to altered protein levels or the creation of dominant negatives by partial gene loss.

Many of the mutations identified in this study impact NFκB signaling distal to the TLR4/MYD88 pathway. MYBBP1A, mutated in 2 of 30 (7%) patients, is thought to inhibit NFκB activity by repressing RELA.57  Copy loss of HIVEP2 (8 of 10; 80%; 23 of 30 in validation) and TNFAIP3 in 5 of 10 (50%) of the paired patients is of interest since their loss results in the removal of NFκB-negative regulators in WM.12,58  Chromosome 6q is often deleted in WM patients, and the apparent loss of HIVEP2 in 6q-intact patients is particularly compelling as a role for this gene in the pathogenesis of WM. Likewise, BIRC3 loss in 4 of 10 (40%) patients is associated with splenic marginal zone lymphoma and can increase activation of noncanonical NFκB signaling.59  Interestingly, both BIRC3 partner proteins, TRAF2 and TRAF3, that regulate noncanonical NFκB signaling were each found to be mutated in 1 of 30 (3%) patients. Biallelic loss of TRAF3 has been reported by Braggio et al12  in WM patients. These findings may therefore provide an impetus for studying noncanonical NFκB signaling in WM.

The loss in 7 of 10 (70%; 18 of 30 in validation) paired patients of LYN, a kinase that plays a regulatory role for B-cell receptor signaling, along with mutations in CD79B (2 of 30; 7%), indicates a possible role for B-cell receptor signaling in this disease.60,-62  The loss of IBTK in 4 of 10 (40%) patients and the interaction between activated MYD88 and BTK, raises the possibility for BTK-mediated cross-talk with the TLR/MYD88 pathway wherein BTK plays an important role in NFκB activation.28,63,-65  Moreover, CBLB, a gene disrupted in chronic myelogenous leukemia and lost in 5 of 10 (50%) of the paired patients, has been shown to inhibit the TLR4 response during inflammation by controlling TRL4 and MYD88 association, and subsequent NFκB activation.66,67  However, there is still limited data regarding the structure of the MYD88 L265P mutant protein. Signaling pathways that are well documented in the WT setting will need to be carefully re-examined to clarify the contribution of the L265P mutation to downstream MYD88 signaling.

Our WGS studies in WM patients have therefore identified mutations in genes involving TLR, NFκB, and CXCR4 signaling, chromatin remodeling, and cell-cycle regulation. These findings may denote a multistep process for WM evolution from IgM MGUS to asymptomatic and symptomatic WM, which invariably will require comparative, and even prospective longitudinal sequencing studies. These efforts are particularly warranted since the principal mutation (MYD88 L265P) identified in these studies is also present in 50% to 80% of IgM MGUS patients using AS-PCR, signifying its role as an early oncogenic event.10,68,69  Therefore, other mutations are likely to be acquired in the evolution of MGUS to symptomatic WM, and may offer the opportunity to identify those patients at high risk for disease evolution. Lastly, these studies have also identified novel targets for a rational approach to WM treatment, including potentially, the use of inhibitors targeting TLR and CXCR4 signaling.

Presented in abstract form at the 48th annual meeting of the American Society of Clinical Oncology, Chicago, IL, June 1, 2012.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

The authors thank Yaoyu Wang and John Quackenbush at the Center for Cancer Computational Biology at the Dana-Farber Cancer Institute for their assistance in developing the copy number analysis.

This work was supported by Peter S. Bing, the International Waldenström’s Macroglobulinemia Foundation, the Coyote Fund for Waldenström’s Macroglobulinemia, the D’Amato Family Fund for Genomic Discovery, the Edward and Linda Nelson Fund for Waldenström’s Macroglobulinemia Research, and the WM patients who provided their samples.

Contribution: Z.R.H. and S.P.T. designed the study and wrote the manuscript; Z.R.H. performed the data analysis and conducted the copy number validation; Z.R.H. and X.L. designed Sanger sequencing primers; G.Y., Y.Z., Y.C., X.L., and L.X. prepared the samples and performed the Sanger validation studies; S.P.T. and P.S. provided patient care, and obtained consent and samples; and R.J.M., C.T., and C.J.P. selected samples and provided clinical data analysis.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Steven P. Treon, Bing Center for Waldenström’s Macroglobulinemia, Dana-Farber Cancer Institute, M547, 450 Brookline Ave, Boston, MA 02215; e-mail: steven_treon@dfci.harvard.edu.

1
Kyle
RA
Therneau
TM
Rajkumar
SV
et al
Long-term follow-up of IgM monoclonal gammopathy of undetermined significance.
Blood
2003
102
10
3759
3764
2
Baldini
L
Goldaniga
M
Guffanti
A
et al
Immunoglobulin M monoclonal gammopathies of undetermined significance and indolent Waldenstrom’s macroglobulinemia recognize the same determinants of evolution into symptomatic lymphoid disorders: proposal for a common prognostic scoring system.
J Clin Oncol
2005
23
21
4662
4668
3
Morra
E
Cesana
C
Klersy
C
et al
Clinical characteristics and factors predicting evolution of asymptomatic IgM monoclonal gammopathies and IgM-related disorders.
Leukemia
2004
18
9
1512
1517
4
Treon
SP
Xu
L
Yang
G
et al
MYD88 L265P somatic mutation in Waldenström’s macroglobulinemia.
N Engl J Med
2012
367
9
826
833
5
Chapman
MA
Lawrence
MS
Keats
JJ
et al
Initial genome sequencing and analysis of multiple myeloma.
Nature
2011
471
7339
467
472
6
Puente
XS
Pinyol
M
Quesada
V
et al
Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.
Nature
2011
475
7354
101
105
7
Montesinos-Rongen
M
Godlewska
E
Brunn
A
Wiestler
OD
Siebert
R
Deckert
M
Activating L265P mutations of the MYD88 gene are common in primary central nervous system lymphoma.
Acta Neuropathol
2011
122
6
791
792
8
Ngo
VN
Young
RM
Schmitz
R
et al
Oncogenically active MYD88 mutations in human lymphoma.
Nature
2011
470
7332
115
119
9
Pasqualucci
L
Trifonov
V
Fabbri
G
et al
Analysis of the coding genome of diffuse large B-cell lymphoma.
Nat Genet
2011
43
9
830
837
10
Jiménez
C
Sebastián
E
Chillón
MC
et al
MYD88 L265P is a marker highly characteristic of, but not restricted to, Waldenström’s macroglobulinemia.
Leukemia
2013
27
8
1722
1728
11
Treon
SP
Hunter
ZR
Aggarwal
A
et al
Characterization of familial Waldenstrom’s macroglobulinemia.
Ann Oncol
2006
17
3
488
494
12
Braggio
E
Keats
JJ
Leleu
X
et al
Identification of copy number abnormalities and inactivating mutations in two negative regulators of nuclear factor-kappaB signaling pathways in Waldenstrom’s macroglobulinemia.
Cancer Res
2009
69
8
3579
3588
13
Schop
RFJ
Van Wier
SA
Xu
R
et al
6q deletion discriminates Waldenström macroglobulinemia from IgM monoclonal gammopathy of undetermined significance.
Cancer Genet Cytogenet
2006
169
2
150
153
14
Braggio
E
Keats
JJ
Leleu
X
et al
High-resolution genomic analysis in Waldenström’s macroglobulinemia identifies disease-specific and common abnormalities with marginal zone lymphomas.
Clin Lymphoma Myeloma
2009
9
1
39
42
15
Nguyen-Khac
F
Lambert
J
Chapiro
E
et al
Groupe Français d’Etude de la Leucémie Lymphoïde Chronique et Maladie de Waldenström (GFCLL/MW); Groupe Ouest-Est d’étude des Leucémie Aiguës et Autres Maladies du Sang (GOELAMS); Groupe d’Etude des Lymphomes de l’Adulte (GELA)
Chromosomal aberrations and their prognostic value in a series of 174 untreated patients with Waldenström’s macroglobulinemia.
Haematologica
2013
98
4
649
654
16
Owen
RG
Treon
SP
Al-Katib
A
et al
Clinicopathological definition of Waldenstrom’s macroglobulinemia: consensus panel recommendations from the Second International Workshop on Waldenstrom’s Macroglobulinemia.
Semin Oncol
2003
30
2
110
115
17
Futreal
PA
Coin
L
Marshall
M
et al
A census of human cancer genes.
Nat Rev Cancer
2004
4
3
177
183
18
Morin
RD
Mendez-Lago
M
Mungall
AJ
et al
Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.
Nature
2011
476
7360
298
303
19
Ngo
HT
Leleu
X
Lee
J
et al
SDF-1/CXCR4 and VLA-4 interaction regulates homing in Waldenstrom macroglobulinemia.
Blood
2008
112
1
150
158
20
Hernandez
PA
Gorlin
RJ
Lukens
JN
et al
Mutations in the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined immunodeficiency disease.
Nat Genet
2003
34
1
70
74
21
Balabanian
K
Lagane
B
Pablos
JL
et al
WHIM syndromes with different genetic anomalies are accounted for by impaired CXCR4 desensitization to CXCL12.
Blood
2005
105
6
2449
2457
22
Wu
B
Chien
EYT
Mol
CD
et al
Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists.
Science
2010
330
6007
1066
1071
23
Orsini
MJ
Parent
JL
Mundell
SJ
Marchese
A
Benovic
JL
Trafficking of the HIV coreceptor CXCR4. Role of arrestins and identification of residues in the c-terminal tail that mediate receptor internalization [published correction appears in J Biol Chem. 2000;275(33):25876].
J Biol Chem
1999
274
43
31076
31086
24
Haribabu
B
Richardson
RM
Fisher
I
et al
Regulation of human chemokine receptors CXCR4. Role of phosphorylation in desensitization and internalization.
J Biol Chem
1997
272
45
28726
28731
25
Lagane
B
Chow
KYC
Balabanian
K
et al
CXCR4 dimerization and beta-arrestin-mediated signaling account for the enhanced chemotaxis to CXCL12 in WHIM syndrome.
Blood
2008
112
1
34
44
26
Poulain
S
Roumier
C
Decambron
A
et al
MYD88 L265P mutation in Waldenstrom macroglobulinemia.
Blood
2013
121
22
4504
4511
27
Leleu
X
Eeckhoute
J
Jia
X
et al
Targeting NF-kappaB in Waldenstrom macroglobulinemia.
Blood
2008
111
10
5068
5077
28
Yang
G
Zhou
Y
Liu
X
et al
A mutation in MYD88 (L265P) supports the survival of lymphoplasmacytic cells by activation of Bruton tyrosine kinase in Waldenström macroglobulinemia.
Blood
2013
122
7
1222
1232
29
Dale
DC
Bolyard
AA
Kelley
ML
et al
The CXCR4 antagonist plerixafor is a potential therapy for myelokathexis, WHIM syndrome.
Blood
2011
118
18
4963
4966
30
Poulain
S
Ertault
M
Leleu
X
et al
SDF1/CXCL12 (-801GA) polymorphism is a prognostic factor aftertreatment initiation in Waldenstrom macroglobulinemia.
Leuk Res
2009
33
9
1204
1207
31
Hooks
SB
Callihan
P
Altman
MK
Hurst
JH
Ali
MW
Murph
MM
Regulators of G-Protein signaling RGS10 and RGS17 regulate chemoresistance in ovarian cancer cells.
Mol Cancer
2010
9
289
32
Busillo
JM
Benovic
JL
Regulation of CXCR4 signaling.
Biochim Biophys Acta
2007
1768
4
952
963
33
Nagl
NG
Jr
Wang
X
Patsialou
A
Van Scoy
M
Moran
E
Distinct mammalian SWI/SNF chromatin remodeling complexes with opposing roles in cell-cycle control.
EMBO J
2007
26
3
752
763
34
Choi
J
Ko
M
Jeon
S
et al
The SWI/SNF-like BAF complex is essential for early B cell development.
J Immunol
2012
188
8
3791
3803
35
Heltemes-Harris
LM
Willette
MJL
Ramsey
LB
et al
Ebf1 or Pax5 haploinsufficiency synergizes with STAT5 activation to initiate acute lymphoblastic leukemia.
J Exp Med
2011
208
6
1135
1149
36
Jones
S
Li
M
Parsons
DW
et al
Somatic mutations in the chromatin remodeling gene ARID1A occur in several tumor types.
Hum Mutat
2012
33
1
100
103
37
Guan
B
Wang
T-L
Shih
IM
ARID1A, a factor that promotes formation of SWI/SNF-mediated chromatin remodeling, is a tumor suppressor in gynecologic cancers.
Cancer Res
2011
71
21
6718
6727
38
Inoue
H
Giannakopoulos
S
Parkhurst
CN
et al
Target genes of the largest human SWI/SNF complex subunit control cell growth.
Biochem J
2011
434
1
83
92
39
Humbert
N
Martien
S
Augert
A
et al
A genetic screen identifies topoisomerase 1 as a regulator of senescence.
Cancer Res
2009
69
10
4101
4106
40
Shadat
NMA
Koide
N
Khuda
II-EI-E
et al
Retinoblastoma protein-interacting zinc finger 1 (RIZ1) regulates the proliferation of monocytic leukemia cells via activation of p53.
Cancer Invest
2010
28
8
806
812
41
Wall
M
Rayeroux
KC
MacKinnon
RN
Zordan
A
Campbell
LJ
ETV6 deletion is a common additional abnormality in patients with myelodysplastic syndromes or acute myeloid leukemia and monosomy 7.
Haematologica
2012
97
12
1933
1936
42
Stegmaier
K
Pendse
S
Barker
GF
et al
Frequent loss of heterozygosity at the TEL gene locus in acute lymphoblastic leukemia of childhood.
Blood
1995
86
1
38
44
43
Kuiper
RP
Schoenmakers
EF
van Reijmersdal
SV
et al
High-resolution genomic profiling of childhood ALL reveals novel recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell cycle progression.
Leukemia
2007
21
6
1258
1266
44
Bohlander
SK
ETV6: a versatile player in leukemogenesis.
Semin Cancer Biol
2005
15
3
162
174
45
Obrador-Hevia
A
Serra-Sitjar
M
Rodríguez
J
Villalonga
P
Fernández de Mattos
S
The tumour suppressor FOXO3 is a key regulator of mantle cell lymphoma proliferation and survival.
Br J Haematol
2012
156
3
334
345
46
Ticchioni
M
Essafi
M
Jeandel
PY
et al
Homeostatic chemokines increase survival of B-chronic lymphocytic leukemia cells through inactivation of transcription factor FOXO3a.
Oncogene
2007
26
50
7081
7091
47
Karube
K
Nakagawa
M
Tsuzuki
S
et al
Identification of FOXO3 and PRDM1 as tumor-suppressor gene candidates in NK-cell neoplasms by genomic and functional analyses.
Blood
2011
118
12
3195
3204
48
Bakker
WJ
Blázquez-Domingo
M
Kolbus
A
et al
FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1.
J Cell Biol
2004
164
2
175
184
49
Rouault
JP
Rimokh
R
Tessa
C
et al
BTG1, a member of a new family of antiproliferative genes.
EMBO J
1992
11
4
1663
1670
50
Lohr
JG
Stojanov
P
Lawrence
MS
et al
Discovery and prioritization of somatic mutations in diffuse large B-cell lymphoma (DLBCL) by whole-exome sequencing.
Proc Natl Acad Sci U S A
2012
109
10
3879
3884
51
Mullighan
CG
Goorha
S
Radtke
I
et al
Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia.
Nature
2007
446
7137
758
764
52
Waanders
E
Scheijen
B
van der Meer
LT
et al
The origin and nature of tightly clustered BTG1 deletions in precursor B-cell acute lymphoblastic leukemia support a model of multiclonal evolution.
PLoS Genet
2012
8
2
e1002533
53
van Galen
JC
Kuiper
RP
van Emst
L
et al
BTG1 regulates glucocorticoid receptor autoinduction in acute lymphoblastic leukemia.
Blood
2010
115
23
4810
4819
54
Treon
SP
How I treat Waldenström macroglobulinemia.
Blood
2009
114
12
2375
2385
55
Moritake
H
Shimonodan
H
Marutsuka
K
Kamimura
S
Kojima
H
Nunoi
H
C-MYC rearrangement may induce an aggressive phenotype in anaplastic lymphoma kinase positive anaplastic large cell lymphoma: identification of a novel fusion gene ALO17/C-MYC.
Am J Hematol
2011
86
1
75
78
56
Cools
J
Wlodarska
I
Somers
R
et al
Identification of novel fusion partners of ALK, the anaplastic lymphoma kinase, in anaplastic large-cell lymphoma and inflammatory myofibroblastic tumor.
Genes Chromosomes Cancer
2002
34
4
354
362
57
Owen
HR
Elser
M
Cheung
E
Gersbach
M
Kraus
WL
Hottiger
MO
MYBBP1a is a novel repressor of NF-kappaB.
J Mol Biol
2007
366
3
725
736
58
Iwashita
Y
Fukuchi
N
Waki
M
Hayashi
K
Tahira
T
Genome-wide repression of NF-κB target genes by transcription factor MIBP1 and its modulation by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase.
J Biol Chem
2012
287
13
9887
9900
59
Rossi
D
Deaglio
S
Dominguez-Sola
D
et al
Alteration of BIRC3 and multiple other NF-κB pathway genes in splenic marginal zone lymphoma.
Blood
2011
118
18
4930
4934
60
Craxton
A
Jiang
A
Kurosaki
T
Clark
EA
Syk and Bruton’s tyrosine kinase are required for B cell antigen receptor-mediated activation of the kinase Akt.
J Biol Chem
1999
274
43
30644
30650
61
Niiro
H
Allam
A
Stoddart
A
Brodsky
FM
Marshall
AJ
Clark
EA
The B lymphocyte adaptor molecule of 32 kilodaltons (Bam32) regulates B cell antigen receptor internalization.
J Immunol
2004
173
9
5601
5609
62
Gauld
SB
Cambier
JC
Src-family kinases in B-cell development and signaling.
Oncogene
2004
23
48
8001
8006
63
Liu
W
Quinto
I
Chen
X
et al
Direct inhibition of Bruton’s tyrosine kinase by IBtk, a Btk-binding protein.
Nat Immunol
2001
2
10
939
946
64
Jefferies
CA
Doyle
S
Brunner
C
et al
Bruton’s tyrosine kinase is a Toll/interleukin-1 receptor domain-binding protein that participates in nuclear factor kappaB activation by Toll-like receptor 4.
J Biol Chem
2003
278
28
26258
26264
65
Liu
X
Zhan
Z
Li
D
et al
Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk.
Nat Immunol
2011
12
5
416
424
66
Makishima
H
Jankowska
AM
McDevitt
MA
et al
CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations and additional chromosomal aberrations constitute molecular events in chronic myelogenous leukemia.
Blood
2011
117
21
e198
e206
67
Bachmaier
K
Toya
S
Gao
X
et al
E3 ubiquitin ligase Cblb regulates the acute inflammatory response underlying lung injury.
Nat Med
2007
13
8
920
926
68
Xu
L
Hunter
ZR
Yang
G
et al
MYD88 L265P in Waldenstrom’s macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction [published correction appears in Blood. 2013;121(26):5259].
Blood
2013
121
11
2051
2058
69
Varettoni
M
Arcaini
L
Zibellini
S
et al
Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstrom’s macroglobulinemia and related lymphoid neoplasms.
Blood
2013
121
13
2522
2528
Sign in via your Institution